ab201734 - 8-hydroxy 2
deoxyguanosine ELISA
Kit
Instructions for Use
For the competitive quantitative measurement of 8-hydroxy 2
deoxyguanosine (8-OH-dG) in urine, cell culture, plasma and other
sample matrices.
This product is for research use only and is not intended for diagnostic
use.
Version 2 Last Updated 11 September 2015
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
4.
5.
6.
7.
8.
PRECAUTIONS
STORAGE AND STABILITY
MATERIALS SUPPLIED
MATERIALS REQUIRED, NOT SUPPLIED
LIMITATIONS
TECHNICAL HINTS
4
4
4
5
5
6
ASSAY PREPARATION
9.
10.
11.
12.
REAGENT PREPARATION
STANDARD PREPARATION
SAMPLE PREPARATION AND STORAGE
PLATE PREPARATION
7
8
9
13
ASSAY PROCEDURE
13.
ASSAY PROCEDURE
14
DATA ANALYSIS
14.
15.
16.
CALCULATIONS
TYPICAL DATA
ASSAY SPECIFICITY
17
18
19
RESOURCES
17.
18.
TROUBLESHOOTING
NOTES
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20
21
1
INTRODUCTION
1. BACKGROUND
Abcam’s 8-hydroxy 2 deoxyguanosine (8-OH-dG) in vitro ELISA Kit
(ab201734) is designed for the competitive quantitative measurement
of 8-OH-dG in urine, cell culture, plasma and other sample matrices.
Thi ELISA utilizes an 8-hydroxy-2-deoxy Guanosine-coated plate and
an HRP-conjugated antibody for detection which allows for an assay
range of 0.94 - 60 ng/mL, with a sensitivity of 0.59 ng/mL. The other
highlights of this kit are a quick incubation time of 60 minutes, stable
reagents, and an easy to use protocol.
It is important to note that the 8-OHdG antibody used in this assay
recognizes both free 8-OHdG and DNA-incorporated 8-OHdG. Since
complex samples such as plasma, cell lysates, and tissues are
comprised of mixtures of DNA fragments and free 8-OHdG,
concentrations of 8-OHdG reported by ELISA methodology will not
coincide with those reported by LC-MS where the single nucleoside is
typically measured. This should be kept in mind when analyzing and
interpreting experimental results.
8-hydroxy-2-deoxy Guanosine (8-OHdG) is produced by the oxidative
damage of DNA by reactive oxygen and nitrogen species and is an
established marker of oxidative stress. Hydroxylation of guanosine
occurs in response to both normal metabolic processes and a variety
of environmental factors (i.e., any biological process which increases
reactive oxygen and nitrogen species). Increased levels of 8-OHdG are
associated with aging as well as with a number of pathological
conditions including cancer, diabetes, and hypertension.
In complex samples such as plasma, cell lysates, and tissues, 8-OHdG
can exist as either the free nucleoside or incorporated in DNA. Once
the blood enters the kidney, free 8-OHdG is readily filtered into the
urine, while larger DNA fragments remain in the bloodstream. Because
of the complexity of plasma samples, urine is a more suitable matrix for
the measurement of free 8-OHdG than plasma. Urinary levels of 8OHdG range between 2.7- 13 ng/mg creatine, while plasma levels of
free 8-OHdG have been reported to be between 4-21 pg/mL as
determined by LC-MS.
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2
INTRODUCTION
2. ASSAY SUMMARY
Remove appropriate number of
antibody
coated
well
strips.
Equilibrate all reagents to room
temperature.
Prepare
all
the
reagents, standards and samples as
instructed. Plates are pre-coated with
8-OHdG.
Add HRP conjugated 8-OHdG,
antibody and either standard or
sample to appropriate wells.
Wash to
reagents.
remove
all
unbound
Develop the well with TMB
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3
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
All reagents should be stored at -20°C. For optimum storage, the 8OHdG Standard should be aliquotted into smaller portions and then
stored at -20oC. Avoid repeated freeze/thaw cycles (10 μL of Standard
can prepare a triplicate standard curve). Refer to list of materials
supplied for storage conditions of individual components. Note the
storage conditions for individual prepared components in the Reagent
Preparation section.
5. MATERIALS SUPPLIED
Item
8-hydroxy-2-deoxyguanosine: BSA Coated Plate
8-hydroxy-2-deoxyguanosine Standard (Stock)
8-hydroxy-2-deoxyguanosine HRP Conjugated
Monoclonal Antibody
Sample and Standard Diluent (Red)
8-hydroxy-2-deoxyguanosine Antibody Diluent
(Blue)
Wash Buffer Concentrate (10X)
TMB Substrate
Stop Solution
Plate Cover
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Quantity
96 wells
1 vial
(100 μL)
1 vial
(75 μL)
1 vial
(50 mL)
1 vial
(13 mL)
1 vial
(50 mL)
1 vial
(13 mL)
1 vial
(13 mL)
2
Storage
Condition
(Before
Preparation)
-20ºC
-20ºC
-20ºC
-20ºC
-20ºC
-20ºC
-20ºC
-20ºC
-20ºC
4
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

A plate reader capable of measuring absorbance at 450 nm.

Adjustable pipettes and a repeat pipettor.

A source of ‘UltraPure’ water. Water used to prepare all reagents
and buffers must be deionized and free of trace organic
contaminants (‘UltraPure’). Use activated carbon filter cartridges or
other organic scavengers. Glass distilled water (even if double
distilled), HPLC-grade water, and sterile water (for injections) are
not adequate for this assay.

Materials used for Sample Preparation
7. LIMITATIONS

Exact conditions may vary from assay to assay, a standard curve
should be generated for every assay performed.

Bacterial or fungal contamination of either samples or reagents or
cross-contamination between reagents may cause erroneous
results.

Disposable pipette tips, flasks or glassware are preferred, reusable
glassware must be washed and thoroughly rinsed of all detergents
before use.

Improper or insufficient washing at any stage of the procedure will
result in either false positive or false negative results. Completely
empty wells before dispensing fresh 1X Wash Buffer. Do not allow
wells to sit uncovered or dry for extended periods.
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5
GENERAL INFORMATION
8. TECHNICAL HINTS

Kit components should be stored as indicated. All the reagents
should be equilibrated to room temperature before use.

Use a clean disposable plastic pipette tip for each reagent,
standard, or specimen addition in order to avoid crosscontamination.

Thoroughly mix the reagents and samples before use by agitation
or swirling.

All residual washing liquid must be drained from the wells by
efficient aspiration or by decantation followed by tapping the plate
forcefully on absorbent paper. Never insert absorbent paper
directly into the wells.

When pipetting reagents, maintain a consistent order of addition
from well-to-well. This will ensure equal incubation times for all
wells.

Use a new adhesive plate cover for each incubation step.

Minimize lag time between wash steps to ensure the plate does
not become completely dry during the assay.

The use of assay reagents not provided in this kit or amendments
to the protocol can compromise the performance of this assay.

The components in each kit lot number have been quality assured
and warranted in this specific combination only; please do not mix
them with components from other kit lot numbers.

If samples generate greater values than the highest standard, the
samples should be re-assayed at a higher sample dilution.
Similarly, if samples generate lower values than the lowest
standard, the samples should be re- assayed at a lower sample
dilution.

This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that contain
sample, control or standard will vary by product. Review the
protocol completely to confirm this kit meets your
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6
GENERAL INFORMATION
requirements. Please contact our Scientific Support staff with
any questions.
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7
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents and samples to room temperature (18-25°C)
prior to use.
Note: Water used to prepare all reagents and buffers must be
deionized and free of trace organic contaminants (‘UltraPure’). Use
activated carbon filter cartridges or other organic scavengers. Glass
distilled water (even if double distilled), HPLC-grade water, and sterile
water (for injections) are not adequate for this assay.
9.1
1X Wash Buffer
Prepare 1X Wash buffer by diluting 10X Wash Buffer in
distilled or deionized water.
For example, if preparing 500 mL of 1X Wash Buffer, dilute
50 mL of 10X Wash Buffer into 450 mL of distilled water. Mix
well.
Store reconstituted 1X Wash Buffer at +2-8ºC for up to one
(1) month. Do not use 1X Wash Buffer if it becomes visibly
contaminated during storage.
9.2
1X
8-hydroxy-2-deoxyguanosine:
Monoclonal Antibody
HRP
Conjugate
Determine the amount of Antibody Preparation required. For
every strip-well used (8-wells), prepare 0.5 mL of Antibody
Preparation.
Prepare Antibody Preparation by diluting the 8-hydroxy-2deoxyguanosine: HRP Conjugate Antibody Concentrate
1:100 with 8-hydroxy-2-deoxyguanosine Antibody Diluent.
For example, if 6 mL of Antibody Preparation is required
(one whole plate), dilute 60 μL of Antibody in 6 mL of 8hydroxy-2-deoxyguanosine Antibody Diluent. Mix well prior
to use.
9.3
TMB and stop solution have been provided at working
strength.
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8
ASSAY PREPARATION
10. STANDARD PREPARATION

Note: The Standard should be aliquotted into smaller portions before use
to ensure product integrity. Avoid freeze/thaw cycles. (10 μL of Standard
can prepare a triplicate standard curve)
10.1
Centrifuge the 8-hydroxy-2-deoxyguanosine Standard
(Stock) vial before removing the cap. This process with
assure that all of the standard is collected and available.
10.2
Label eight tubes with Standards #1 – 8.
10.3
Add 500 μL of Sample and Standard Diluent to Tube #1.
10.4
Add 250 μL of Sample and Standard Diluent to Tube #2 #7.
10.5
Add 500 μL of Sample and Standard Diluent to Tube #8.
10.6
Add 10 μL of the 3.06 μg/mL 8-hydroxy-2-deoxyguanosine
Standard to Tube #1 for a concentration of 60 ng/mL. Mix
well.
10.7
Transfer 250 μL from Tube #1 to Tube #2. Mix well.
10.8
Using the table below as a guide, repeat for Tubes #4 - #7.
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9
ASSAY PREPARATION
Standard Dilution Preparation Table
Standard
#
Sample to
Dilute
Volume to
Dilute
(µL)
1
Stock
10
Volume
of
Diluent
(µL)
500
2
Standard #1
250
3
Standard #2
250
4
Standard #3
5
6
7
8
Starting
Conc.
(ng/mL)
Final
Conc.
(ng/mL)
3060
60
250
60
30
250
30
15
250
250
15
7.5
Standard #4
250
250
7.5
3.75
Standard #5
250
250
3.75
1.875
Standard #6
Diluent
250
0
250
500
1.875
0
0.94
0
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10
ASSAY PREPARATION
11. SAMPLE PREPARATION AND STORAGE

Urine, cell culture, plasma are suitable sample matrices for use in
the assay.

Proper sample storage and preparation are essential for consistent
and accurate results. Please read this section thoroughly before
beginning the assay.

General Precautions: All samples must be free of organic solvents
prior to assay. Samples that cannot be assayed immediately
should be stored as indicated below. Please be advised that all
suggested dilutions below are simply recommended as a starting
point, and it may be necessary to adjust the dilution based on
experimental results.
11.1. Plasma/Serum
The concentration of free 8-OHdG in plasma is very low
relative to the level of DNA-incorporated 8-OHdG.
Glomerular filtration results in excretion of 8-OHdG into the
urine, while the DNA-incorporated 8-OHdG remains in the
blood. The differing fates of free versus DNA-incorporated 8OHdG should be considered in experimental design. If you
choose to measure DNA-incorporated 8-OHdG in plasma, it
may be preferable to purify DNA using a commercially
available kit and treat the DNA with a combination of
nuclease and alkaline phosphatase to liberate the individual
bases. Due to the complexities of measuring 8-OHdG in
plasma, urine is often a more appropriate matrix.
Storage: Collect plasma using established methods and store
at -80°C.
Dilution: Serum samples may be diluted 1:20 (v:v) in Sample
and Standard Diluent as the starting dilution prior to testing.
11.2. Cell Lysates
Storage: Collect lysates using established methods and store
at -80°C until use.
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11
ASSAY PREPARATION
Usage: Purify DNA using a commercially available extraction
kit. Digest DNA using nuclease P1 following the
manufacturer’s instructions. Adjust pH to 7.5 - 8.5 using 1M
Tris. Add 1 unit of alkaline phosphatase per 100 μg of DNA
and incubate at 37°C for 30 minutes. Boil for 10 minutes and
place on ice until use.
11.3. Urine
Interference in urine is infrequent; dilutions appropriate for
this assay show a direct linear correlation between 8-OHdG
immunoreactivity and 8-OHdG concentration (see figure 1).
Urinary concentrations of 8-OH-dG can vary considerably
and can be standardized against creatinine levels if required.
Storage: Fresh urine samples should be centrifuged at 2,000
x g for 10 minutes or filtered with a 0.2μm filter before this
assay, and stored at -20°C immediately after collection.
Dilution: Dilute urine samples 1:20 (v:v) in Sample and
Standard Diluent as the starting dilution prior to testing. For
example: 9 μL of sample into 171 μL of Sample and
Standard Diluent.
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12
ASSAY PREPARATION
Figure 1. Recovery of 8-hydroxy-2-deoxy Guanosine from urine
Urine samples were spiked with 8-OHdG, diluted as described in the Sample
Preparation section and analyzed using the 8-OHdG ELISA Kit. The yintercept corresponds to the amount of 8-OHdG in un-spiked urine. Error bars
represent standard deviations obtained from multiple dilutions of each sample.
11.4. Culture Media Samples
Storage: Collect culture media samples and store at -80°C.
Dilution: Foetal bovine serum contains 8-OHdG, therefore
assays should either be performed in serum-free medium or
PBS if these samples are assayed directly. If the 8-OHdG
concentration is high enough to dilute the sample 10-fold with
Sample and Standard Diluent, the assay can be performed
without any modifications. When assaying less concentrated
samples (where samples cannot be diluted 1:10 with Sample
and Standard Diluent), dilute the standards in the same
culture medium as that used for the experiment. This will
ensure that the matrix for the standards is comparable to the
samples. We recommend that a standard curve be run first to
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13
ASSAY PREPARATION
ensure that the assay will perform in a particular culture
medium.
11.5. Tissue Samples
Storage: Snap-freeze tissue samples in liquid nitrogen
immediately after collection. Store at -80°C until use.
Usage: When ready to use the samples, thaw and add 5 mL
of homogenization buffer (0.1 M phosphate buffer, pH 7.4,
containing 1 mM EDTA) per gram of tissue. Homogenize the
sample using either a Polytron-type homogenizer or a
sonicator. Centrifuge at 1,000 x g for 10 minutes and purify
the supernatant using a commercially available DNA
extraction kit. Digest DNA using nuclease P1 following the
manufacturer’s instructions. Adjust the pH to 7.5-8.5 using 1
M Tris. Add 1 unit of alkaline phosphatase per 100 μg of DNA
and incubate at 37°C for 30 minutes. Boil for 10 minutes and
place on ice until use.
11.6. Saliva
Storage: Saliva samples should be stored at -80°C
immediately after collection. Samples may be assayed
directly after appropriate dilution.
Dilution: Saliva samples can be prepared 1:8 (v:v) in Sample
and Standard Diluent as a suggested starting dilution.
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14
ASSAY PREPARATION
12. PLATE PREPARATION



The 96-well plate(s) included with this kit is supplied ready to use.
It is not necessary to rinse the plate(s) prior to adding the
reagents.
NOTE: If you do not need to use all the strips at once, place the
unused strips back in the plate packet and store at +2-4°C. Be
sure the packet is sealed with the desiccant inside.
For statistical purposes, we recommend assaying samples in
triplicate.
A suggested plate format is shown in Figure 2, below. The user
may vary the location and type of wells present as necessary for
each particular experiment. The plate format provided below has
been designed to allow for easy data analysis.
Figure 2. Sample plate format. Blk (Blank), S1-S8 (Standards 1-8),
1-24 (Samples)
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15
ASSAY PROCEDURE
13. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room
temperature prior to use.

Pipetting Hints: Use different tips to pipette the buffer,
standard, sample, tracer, and antibody. Before pipetting each
reagent, equilibrate the pipette tip in that reagent (i.e., slowly
fill the tip and gently expel the contents, repeat several times).
Do not expose the pipette tip to the reagent(s) already in the
well.
Addition of the Reagents (please refer to Pipetting Summary
Table below)
13.1
13.2
Add 50 μL (in triplicate) of each of the following to
appropriate wells:

Prepared 8-hydroxy-2-deoxyguanosine Standard
(Tube #1 - Tube #7) into wells labelled S1-S7

Zero Standard (Tube #8- Sample and Standard
Diluent, which represents 0 ng/mL) into wells
labelled S8

Samples (previously prepared- See Sample
Preparation) into wells labelled 1-23
Add 50 μL of the previously diluted 8-hydroxy-2deoxyguanosine Antibody Preparation to each well, except
the blank.
13.3
Add 50 μL of Standard and Sample Diluent and 50 μL of
Antibody Diluent into wells labelled as the blank.
13.4
Cover each plate with the plate cover and incubate 1 hour
at room temperature (+20- 25˚C).
Carefully remove adhesive plate cover. Gently squeeze the
long sides of the plate frame before washing to ensure all
strips remain securely in the frame.
13.5
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16
ASSAY PROCEDURE
13.6
Empty plate contents. Use a multi-channel pipette or a plate
washer to fill each well completely (300 μL) with 1X Wash
buffer, then aspirate plate contents. Repeat procedure three
additional times, for a total of FOUR washes. Blot plate by
tapping gently onto paper towels or other absorbent
material.
NOTE: Follow the same procedure when using an automated
plate washer as well. Take care to avoid microbial contamination
of equipment. Automated plated washers can easily become
contaminated thereby causing assay variability.
Well
Standard
OR Sample
Preparation
Standard
(S1-S7)
50 μL
Zero
Standard
(S8)
Blank
Samples
(1-23)
Standard
&
Sample
Diluent
Included
in
Standard
Preparati
on
50 μL
50 μL
50 μL
Included
in Sample
Preparati
on
Antibody
Preparation
Antibody
Diluent
Total
Volume/
Well
50 μL
Included in
Antibody
Preparation
100 μL
50 μL
50 μL
Included in
Antibody
Preparation
50 μL
Included in
Antibody
Preparation
100 μL
100 μL
100 μL
Pipetting Summary Table
NOTE: Only remove the required amount of TMB Substrate and
Stop Solution for the number of strips being used. Do NOT use a
glass pipette to measure the TMB Substrate solution. Do NOT
return leftover TMB Substrate to bottle. Do NOT contaminate the
unused TMB Substrate. If the solution is blue before use, DO
NOT USE IT.
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17
ASSAY PROCEDURE
13.7
13.8
13.9
Add 100 μL of TMB Substrate into each well.
Cover carefully with the second provided plate cover.
Allow the enzymatic color reaction to develop at room
temperature (+20-25ºC) in the dark for 30 minutes. The
substrate reaction yields a blue solution.
13.10 After 30 minutes, carefully remove the plate cover, and stop
the reaction by adding 100 μL of Stop Solution to each well.
Tap plate gently to mix. The solution in the wells should
change from blue to yellow.
NOTE: Note: Evaluate the plate within 30 minutes of stopping the
reaction.
13.11 Wipe underside of wells with a lint-free tissue.
13.12 Measure the absorbance on the ELISA plate reader set at
450 nm.
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18
DATA ANALYSIS
14. CALCULATIONS
Many plate readers come with data reduction software that plot data
automatically.
Preparation of the Data
The following procedure is recommended for preparation of the data
prior to graphical analysis.
Note: If the plate reader has not subtracted the absorbance readings of
the blank wells from the absorbance readings of the rest of the plate,
be sure to do that now.
14.1
For each sample and standard, subtract the average blank
absorbance value (OD) and calculate the average for each
of the replicates.
14.2
Plot the average absorbance versus 8-OHdG concentration
of the standards. Sample concentrations may be
extrapolated from the standard curve using the blank
subtracted average absorbance values.
14.3
Samples with absorbance values outside of the standard
curve should be measured again using a dilution which will
bring the absorbance into the range of the standard curve.
Remember to use the dilution factor which calculating the
concentration present in the original sample.
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DATA ANALYSIS
15. TYPICAL DATA
The standard curve presented here (Figure 3 below) is an example of
the data typically produced using this assay; however, your results will
not be identical to these. It is necessary to measure a new standard
curve each time the assay is used. Development conditions and the
purity of the water used can substantially alter your results from the
data presented below.
Figure 3. This standard curve was generated using the 8-OHdG ELISA Kit
Protocol and is typical of the performance of this assay. This standard curve is
for demonstration only. A fresh standard curve must be generated for each
assay.
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DATA ANALYSIS
The intra- and inter-assay CV’s have been determined and can be
seen below.
Sample
numbers
3
Assay
replicates
30
n
90
Intra-assay
variation (CV %)
<5
Inter-assay
variation (CV %)
<5
Table 1. Intra- and inter-assay variation. %CV represents the variation in
concentration (not absorbance) as determined using three samples of known
concentration which were assayed thirty times on one plate (Intra-assay
variation) and 30 times in 3 individual assays (Inter-assay variation).
16. ASSAY SPECIFICITY
Specificity of the 8-hydroxy-2-deoxyguanosine Monoclonal Antibody:
Compound
Cross-reactivity (%)
8-hydroxy-2-deoxyguanosine
100
8-hydroxy guanosine
23
8-hydroxy guanine
23
Guanosine
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<0.01
21
RESOURCES
17. TROUBLESHOOTING
Problem
Possible Causes
Improper standard
solution
Standard degraded
Poor Standard Curve
Curve doesn’t fit scale
Pipetting Error
Plate washings too
vigorous
No Signal
Wells dried out
Target present below
detection levels of kit
High Background
Low sensitivity
Wells are insufficiently
washed
Contaminated wash
buffer
Waiting too long to read
the plate after adding
stop solution
Standard is degraded
Mixing or substituting
reagents from other kits
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Solutions
Confirm dilutions are
made correctly.
Store and handle
standard as
recommended
Try plotting using
different scales
Use calibrated pipettes
and proper pipetting
technique.
Check and ensure
correct pressure in
automatic wash system.
Pipette wash buffer
gently if washes are
done manually.
Do not allow wells to dry
out. Cover the plate for
incubations.
The basic range of DNA
to use, if the damage is
low, 100ug/ml - if
maximally damaged,
1ng/ml and dilute from
there.
Wash wells as per
protocol
Prepare fresh wash
buffer
Read plate immediately
Replace standard
Avoid mixing
components
22
RESOURCES
18. NOTES
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23
UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
Austria
Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
France
Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
Spain
Email: soportecientifico@abcam.com | Tel: 911-146-554
Switzerland
Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226)
Canada
Email: ca.technical@abcam.com | Tel: 877-749-8807
China and Asia Pacific
Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通)
Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
www.abcam.com | www.abcam.cn | www.abcam.co.jp
Copyright © 2015 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
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